We previously isolated cells impaired in general phagocytosis from a macrophage cell line by selecting mutants resistant to killing by toxin-derivitized plastic beads. Several mutants could not phagocytose beads, but were normal in receptor-mediated phagocytosis. We now find that two mutants, unlike the parent, are detached from flasks by trypsin. The defects in the mutants are under study. Differences in virulence among three Salmonella strains were shown by us to result from differential complement (C) activation via the alternative pathway. Only the structure of O-antigen (O-Ag) in their lipopolysaccharide (LPS) differs among these strains; this difference causes the different C activation since the results were mimicked by purified LPS coated on sheep erythrocytes. We now find that during activation, C3b deposition on either bacteria or LPS-coated erythrocytes is proportional to C3 activation, and that the degradation of this surfacebound C3b (e.g., to iC3b) is independent of LPS structure. Thus, slight differences in O-Ag composition affect C3 activation but not its degradation; the resultant covalently bound C3 fragments presumably determine subsequent phagocytosis by macrophages via the approporate receptors, thereby influencing bacterial virulence. Bacteria containing O-Ag are not killed by the terminal attack complex of C (C5b-9) but those lacking O-Ag are. Using a Salmonella mutant in which the synthesis of O-Ag can be varied, we found that sensitivity to killing increased abruptly when less than 60% of the LPS molecules have O-Ag, and less than 20% of the LPS molecules have long (15-55 repeating units) O-Ag chains. Further, two classes of E. coli mutants resistant to C killing were both found to have increased amounts of LPS; one also had increased substitution of LPS with O-Ag. These results, with different organisms, indicate that a specific critical degree of steric hindrance by O-Ag prevents access of the C5b-9 complex to the membrane.